1. Field of the Invention
This invention relates to immunoassay of an analyte and materials relates to immunoasay of an analyte and materials used therein, and more particularly relates to a method and materials for enzyme immunoassay in which the analyte is detected by a colored spot on a solid support.
2. Background of the Invention
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous, requiring a separation of bound tracer from free (unbound) tracer or homogeneous in which a separation step is not required.
Enzymes have often been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a color change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
Peroxidases are commonly used as labels in EIA. These enzymes catalyze the oxidation of a substrate to a product by hydrogen perioxide. Horseradish peroxidase (HRP) is a widely used example of this class of enzymes. U.S. Pat. No. 4,016,043 to Schuurs et al. discloses EIA using HRP.
Numerous substrates oxidized by hydrogen peroxide under catalysis by a peroxidase are known. Among the more common are 3,3'-diaminobenzidine (DAB), 5-amino salicylic acid, o-dianisidine, o-toluidine and, most commonly, o-phenylenediamine (OPD).
These known peroxidase substrates, while useful, have certain limitations. For example, the oxidation product of OPD has sufficient water solubility so that it gives an excellent visual readout in a solution assay. On the other hand, the water solubility of the oxidation product causes rapid diffusion when the product is deposited as a spot on a solid phase, such as a membrane or dipstick. This severely reduces assay sensitivity and limits usefulness of OPD in ELISA procedures.
Several peroxidase substrates which generate insoluble products are known, such as DAB, 3-amino-9-ethylcarbazole, 3,3',5,5'-tetramethyl-benzidine, and 4-chloro-1 naphthol. While effective, these substrates do not generate as much color as is developed with OPD and peroxidase.
Lactoperoxidase (LPO) is a commercially available enzyme from milk which catalyzes the oxidation of iodide to iodine by peroxide. This reaction has been used for preparation of an immunoassay reagent labeled with .sup.127 I in U.S. Pat. No. 4,687,734 to Chester.
U S. Pat. No. 4,504,579 to Sun discloses an antibiotic-stabilized conjugate of LPO and an immunological binding component useful as an EIA reagent.
There is need for a method for peroxidase based EIA which gives a stable, well defined and deeply colored spot on a membrane or dipstick. It is toward fulfillment of this need that this application is directed.